Vitamin D receptor rs3782905 and vitamin D binding protein rs7041 polymorphisms are associated with hepatocellular carcinoma susceptibility in cirrhotic HCV patients

Background The severity of chronic hepatitis C and susceptibility to hepatocellular carcinoma (HCC) are associated with genetic variations within vitamin D receptor (VDR) in several populations. This study aims to determine the significance of the VDRs (rs2228570, rs3782905, rs11568820) and DBP (rs7041) for the susceptibility to HCC in Egyptian patients with chronic HCV infection and their effect on the progression of liver cirrhosis to carcinogenesis. Methods Single nucleotide polymorphisms (SNPs) VDR (rs2228570, rs3782905), and DBP rs7041 were genotyped using restriction fragment length-PCR (RFLP-PCR) technique and VDR rs11568820 was genotyped using single strand polymorphism PCR (SSP PCR). These SNPs genotypes, haplotypes and linkage disequilibrium analyses were examined in 299 Egyptian individuals (100 HCV-cirrhotic patients, 99 HCC- HCV patients, and 100 healthy controls). Result The VDR rs2228570 CC genotype, VDR rs3782905 GC and CC genotypes, and DBP rs7041 GG genotype are significantly higher in HCC. It is noteworthy that, VDR rs3782905 CC and DBP rs7041 TG genotypes are higher in HCV induced liver cirrhosis than with HCC progression in HCV infected patients. Furthermore, among patients, the relationship between these SNPs and smoking status, gender, and HCC susceptibility was reported. Conclusion Among the four investigated SNPs, there are associations between VDR rs3782905 and DBP rs7041 and the HCC progression in Egyptian patients chronically infected with HCV. These SNPs are considered as risk factors in HCV induced liver cirrhosis and HCC. The combinations of these SNPs with smoking status and gender are statistically linked to a high risk of HCC. Future research with a larger sample size of subjects with HCV infection is advised, because chronic liver disease induced by HCV infection is the primary cause of HCC in Egypt. We recommend screening of these SNPs for prediction of LC and HCC development in HCV infected patients, which may improve the used therapeutic protocol. These results suggest that VDR polymorphisms may be potential determinants for HCC susceptibility in Egyptian HCV patients. Supplementary Information The online version contains supplementary material available at 10.1186/s12920-023-01749-8.

authorities in Egypt have reported that HCC is considered the most challenging health problem.Over ten years, the number of HCC patients increased by more than twice ( [3].HCC is a complicated, multistage, and multifaceted process [4,5]).Hepatitis C virus (HCV) infection, a prominent cause of cirrhosis (93%) worldwide [6], is also a risk factor for HCC [7].There are several known risk factors for HCC, including tobacco use, certain environmental carcinogens, and familial or genetic factors [8].
Vitamin D, which is working through binding with VDR, is involved in cancer and inflammatory liver disease development [9].Vitamin D receptors (VDR), both nuclear and cytosolic, are required for vitamin D action in cells.VDR ligands affect the expression of 500-1000 genes [10].Multiple allelic variations exist in the VDR gene located on chromosome 12q.Numerous single nucleotide polymorphisms (SNPs) are studied, some of which are involved in the growth of tumors and chronic liver diseases [11].Fok1, Bsm1, APa1, and Taq1 are the four SNPs that have been studied the most [12].
The vitamin D-binding protein (DBP) is the most crucial transport protein for vitamin D in the blood.The amount of active vitamin D in the blood greatly influances the DBP levels [13].To maintain the physiological level of vitamin D, renal tubular cells must produce, filter, and then reabsorb the DBP/25-hydroxyvitamin D [25(OH) D] via VDRs [14].The highly polymorphic "group-specific component" (GC) gene, which encodes the DBP protein located on chromosome 4 (4q11-13) [15].The two DBP most common polymorphisms, rs7041 (c.1296 T > G) and rs4588 (c.1307C > A), were described as non-synonymous coding SNP [16].
The involvement of VDR SNPs in HCC development was reported in several populations [17][18][19][20][21][22][23] with fluctuating data, but a few VDR SNPs have been examined concerning HCC risk factors in the Egyptian population [24][25][26][27], however, it is still unclear in Egyptian patients whether VDR and DBP SNPs could be risk factors in chronic HCV-infected HCC patients.As a result, this study aims to determine the significance of the VDRs (rs2228570, rs3782905, rs11568820) and DBP (rs7041) for the susceptibility to HCC in Egyptian patients with chronic HCV infection and their effect on the progression of liver cirrhosis to carcinogenesis.

Study participants
Prospectively, the 299 Egyptians were chosen from the inpatient and outpatient clinics of the Tropical Medicine Department of the Mansoura University Hospital in Egypt.The participants were classified into 3 groups: 99 HCV-HCC patients, 100 HCV-cirrhotic patients and 100 individuals as controls with no prior history of liver disease (HBV and HCV Negative).Patients with HCV were diagnosed or had been undergoing follow-up care.The HCV infection was confirmed by anti-HCV antibodies and HCV RNA by using ELISA and Real-time PCR tests.HCC patients were diagnosed by serum alphafetoprotein (AFP) in addition to imaging by abdominal US and spiral CT.Diagnostic imaging standards with high specificity for HCC ≥ 10 mm have been established by the American College of Radiology through its Liver Imaging Reporting and Data System (LIRADS) and previous AASLD guidelines [28].These include hyperenhancement in the arterial phase combined with washout appearance and/or capsule appearance.Lesions that did not meet these guidelines or were smaller than 1 cm were not incorporated into this study.Liver cirrhosis (LC) in patients was diagnosed based on combined clinical, laboratory, and radiological findings.In some unclear cases, certain imaging tests, including transient or magnetic resonance elastography (MRE), CT and ultrasound may be recommended.Those who had HIV or HBV and autoimmune disease were not included in this study.In accordance with the Declaration of Helsinki and with participants' signed informed permission, the local institutional review board of the Faculty of Medicine at Mansoura University accepted our present patient study.

Laboratory analyses
A sample of 5 ml of peripheral blood was taken from each participant.Three ml of the sample were collected in dry tubes to obtain sera for evaluation of biochemical parameters.For molecular analysis, 2 ml of the sample were collected in sterile tubes containing ethylene-diaminetetraacetic acid (EDTA).Full clinical examination and full history were performed on all patients.Baseline measurements of the participants' liver biochemical profile and AFP were made according to the manufacturer's recommendations for each kit.

Molecular analysis DNA isolation
Genomic DNA was extracted using the the Wizard Genomic DNA Integrity Kit according to the manufacturer's instructions (Promega, Madison, USA).The 1% agarose gel was used to demonstrate the DNA's integrity.Using NanoDrop ™ 2000/2000c, the purity and concentration of DNA were assessed (Thermo Fisher Scientific, Waltham, MA, USA).In order to perform SNP genotyping, the extracted DNA is kept at -20 ºC.

Genotyping of VDR and DBP
The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to identify the SNPs of rs2228570, rs3782905, and rs7041 Table 1.PCR was performed in a total volume of 12.5 μl using Biometra II thermal cycler (Göttingen, Germany).The PCR cycles were as follows: 94˚C for 7 min, 30 cycles of denaturation at 95 ˚C for 45 s, annealing at the indicated temperatures in Table 1 for 45 s, extension at 72 ˚C for 45 s, and a final extension at 72 ˚C for 5 min.
The PCR products were visualized under UV on 2% agarose gel, which was stained with ethidium bromide.After amplification, the PCR products were digested for 1 h and then visualized on a 2.5% agarose gel, as described in Table 1.SNP rs11568820 was genotyped by the single-stranded polymorphism polymerase chain reaction (SSP-PCR), was performed with the following cycling program: 94˚C for 7 min, 30 cycles of denaturation at 95 ˚C for 45 s, annealing at 45 o C for 45 s, and extension at 72 ˚C for 45 s, and a final extension at 72 ˚C for 5 min.Annealing temperatures, restriction enzymes, and product size in genotype analysis are summarized in Table 1.The PCR-SSP was performed in a total volume of 12.5 μl reaction.PCR cycles were performed as before in Table 1.

Statistical analysis
Data were computed and statistically analyzed using the SPSS software program (IBM Corp. Released 2012, Version 21.0.IBM SPSS Statistics for Windows; Armonk, NY, USA).In terms of continous variables, the median was expressed along with the 25 th and 75 th percntiles.The non parametric Kruskal-Wallis test and Mann-Whitney U tests were employed to look at variations in demographic traits and laboratory databetween groups.Different genotypes for each SNP were defined in number and minor allele frequencies were calculated.Hardy-Weinberg equilibrium (HWE) was assessed for each SNP to insure that the selected SNPs were informative enough for genetic statistical analysis and the selected groups represent our population (HWE; p-value > 0.05).The risk was indicated by an odds ratio (OR) and a 95% confidence interval (95% CI).Nominal logistic regression models were used to evaluate the association between the independent studied groups and a nominal dependent genotype model.We also used Chi-square (χ 2 ) test to compare qualitative variables in each group separately to check for variations in allele and genotype distribution.Using population genotype data, the SHEsis software (http:// analy sis.bio-x.cn/ myAna lysis.php) performed linkage disequilibrium (LD) construction and haplotype evaluation [29].All results were two-tailed, and statistical significance was set at p < 0.05.The Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) were used to determine the best fit model for each SNP.The inheritance model (codominant, dominant, recessive, and overdominant) of these SNPs was constructed using SNP Stats (https:// www.snpst ats.net/ start.htm).

Demographic and clinical characteristics
The demographic and clinical characteristics of the studied groups are outlined in Table 2.In our study, we included 39 males and 61 females cirrhotic patients with a median age of 50 (42.25-58.75)and patients with HCC were 46 males and 53 females with a median age of 55 (47-60).The age differences between the three groups were statistically significant (p < 0.001), while no Table 1 The sequence of the primers and PCR-RFLP products characteristics Enzyme (BseG I) investigates SNP (rs2228570), Enzyme (HpyF3 I) investigates SNP (rs3782905), Enzyme( BsuR I) investigates SNP (rs7041) significant differencesconcerning gender and smoking were observed.

SNPS
As shown in Table 2, the biochemical characteristics ALT, AST,creatinine, T-Bil, D-Bil, and AFP were significantly higher in HCC patients than in healthy controls and HCV cirrhotic patients (p < 0.001), while ALB was significantly lower (p < 0.001).

VDR and DBP genotypic distribution
Frequencies of the groups' genotypes and alleles are shown in Table 3.The four SNPs in the groups had genotype distributions that fell within the Hardy-Weinberg equilibrium.

VDR (FokI rs2228570) genotypes distribution
The PCR-RFLP targeting rs2228570 was represented in Fig. 1.The genotypic frequency of VDR rs2228570 in HCV cirrhotic and HCC patients had a high prevalence of CC genotype compared to healthy control (OR = 2.279; CI: 1.283-4.033,p < 0.01, OR = 1.685;CI: 0.951-2.985,p < 0.05), respectively.The distribution of genotypes and alleles were insignificant differences by comparing HCC patients to both the healthy control group and the cirrhotic group.

VDR (Cdx-2 rs11568820) genotype distribution
The PCR-SSR targeting rs11568820 was represented in Fig. 3. Regarding rs11568820 polymorphism, we have detected only one genotype in all participants (GA genotype).This genotype and allele frequencies in all studied groups did not differ significantly.

Haplotype analysis
Estimating haplotypes of rs2228570, rs3782905, rs11568820, and rs7041 polymorphisms in all studied groups gave eight haplotypes as shown in     ) 1S and 2S.

The correlation of SNPs to smoking and gender
In non-smoker patients, the genotypic frequency of rs7041 had a high prevalence of the GG genotype (OR = 2.23; CI: 1.07-4.64,p < 0.05).However in smoker patients, the genotypic frequency of rs7041 had a high prevalence of GG genotype (OR = 3.44; CI: 1.28-9.26,p < 0.05).Also, in non -smoker patients the genotypic frequency of DBP rs7041 had a high prevalence of GG genotype (OR = 2.23; CI: 1.07-4.64,p < 0.05), as shown in Table 5.Furthermore, in females the genotypic frequency of DBP rs7041 had a high prevalence of GG genotype (OR = 3.94; CI: 1.80-8.61,p < 0.01).In males the genotypic frequency VDR rs3782905 had also a high prevalence of GC genotype (OR = 2.56; CI: 1.03-6.30,p < 0.05), as shown in Table 6.

Discussion
In chronic hepatitis C patients, the precise process by which HCC develops, taking into account both host and viral elements, remains uncertain [30].The high gender disparity in HCC is caused by differences in how each gender is exposed to the causal agents, as well as genetic factors including inflammatory cytokine and growth factor receptor gene polymorphisms [23,[30][31][32].Finding more genetic factors that affect the transcription of particular regulatory genes could therefore help to identify high-risk groups.Therefore, the current study's goal was to determine whether there was an association between the HCC in cirrhotic HCV patients in the Egyptian population and SNP of VDR (FOKI rs 2228570, BsmI rs 3782905, Cdx-2 rs 11568820) and DBP(rs7041).The overall HCC rate was significantly greater in patients aged 55 and older.This finding is the line with Yi et al. [33], who found that male sex and age > 55 in a Chinese population were linked to a higher risk of Fig. 3 PCR-SSR product of rs11568820 polymorphism, lane (M) 100 bp ladder, lanes (1,16) were GA genotype 297, 235 and 110 bp (croped) Fig. 4 PCR-RFLP product of DBP rs7041 polymorphism, lane (1) 100 bp ladder, lanes (4,7,812,16) were TT genotype 482 bp, lanes (2,5,7,9,10,14,15) were TG genotype 482, 298 and 184 bp, and lanes (1,3,11,13) were GG genotype 298 and 184 bp having HCC.Regarding laboratory data, it was found that the ALT, AST, ALB, creatinine, T-Bil, D-Bil, and AFP levels were significantly different across the three groups.According to Barooah et al. [34] and Raafat et al. [35], they observed higher serum levels of ALT, AST, and bilirubin in HCC patients than in chronic liver disease patients.Additionally, Du et al. [36] observed the risk of HCC in patients with ALT elevation (1.82-2.75-fold).Individuals with chronic HBV who had chronically abnormal ALT may have been included in or referred to by the population that had high ALT.HCC was typically observed in patients with ALT rise, particularly ALT flare (mean 3 months in China).This demonstrates that ALT flare is more likely a predictor than a cause of the development of HCC.Turshudzhyan and Wu [37] also observed an elevation in serum AFP in HCC patients.Several polymorphisms in the VDR genes, including BsmI, FokI, TaqI, and Cdx-2, have been reported in recent years at various loci.These polymorphisms in the VDR gene can change the activity of VDR proteins [11].
There is a research in Nigerian population confirm that some randomized clinical control trials suggesting vitamin D related mechanism is vital in HCC progression [20].In the present study, in both cirrhotic and HCC patients, rs2228570 had a significantly high prevalence of CC genotypes.As a result, the CC genotype may be more than a two-fold factor for liver cirrhosis, and also for a little rise in the risk of HCC.This is in line with Tsounis et al. [38], who reported that the development of cirrhosis is related to homozygosity for the dominant trait of FokI variants.Contrary to Mohammed et al. [26] findings, HCC risk was considerably higher among those with the FokI (TT) genotype.Also, Moemen et al. [27] reported that the homozygosity for the recessive FokI allele (TT) was associated with increased tumor size and higher AFP levels in Egyptian patients with HCV-related HCC.
In this study, we noticed a high prevalence of the TG genotype for rs7041, suggesting that the TG genotype might be a three-fold risk factor for liver cirrhosis compared to healthy controls with significant allelic distributions.The G allele is correlated with an approximately three-fold increase in liver cirrhosis risk.Nevertheless, the GG genotype may be considered a risk factor for HCC and HCV cirrhotic patients because of the high prevalence in HCC and HCV cirrhotic patients.These results came in agreement with Peng et al. [39], who reported that rs7041 GG and the rs7041 TG/GG genotypes 'were correlated with a significantly increased HCC risk in HBV Chinese patients.Also, similar results were reported in Thailand populations by Maneechay et al. [40], which showed that the risk genotypes (TG/GG) in rs7041 were associated with lung cancer.Also, Chupeerach et al. [41] revealed a correlation between colorectal cancer to the same genotypes.
Data on BsmI polymorphism in liver diseases are scanty.The VDR gene polymorphism at rs3782905 in HCV cirrhotic patients exhibited a significant incidence of CC genotype in this study, suggesting that CC genotype might be a risk factor for LC four times higher than healthy controls.The HCC patients had a high prevalence of GC and CC genotypes that might be considered as a risk factor for HCC in comparison with healthy control.The GC genotype increases the risk for the prognosis of LC to HCC.In Egyptian populations, the GC genotype was correlated to a nearly two-fold increase in HCV-related HCC risk.In contrast to Mohamed et al. [26], it was reported that there was no significant association between rs3782905 polymorphism and HCC development.
It is noteworthy that, VDR rs3782905 CC and DBP rs7041 TG genotypes are more associated with a higher risk of HCV-induced liver cirrhosis than with HCC progression in HCV-infected patients.Furthermore, among patients, the relationship between these SNPs and smoking status, gender, and HCC susceptibility was reported.
Looking at the rs11568820 polymorphism, we detected only one genotype in this SNP in all participants (GA genotype).In line with Peng et al. [39], who studied the association between rs11568820 SNP and HBV-related HCC risk in the Chinese population, we found that no significant association between the frequency of alleles and genotypes of rs11568820 polymorphism within the three studied groups with the development of the HCV-related HCC.In contrast, Dai et al. [42] demonstrated that the rs11568820 polymorphism is believed to contribute to HCC carcinogenesis.
Our results indicated that there is no linkage disequilibrium between the 3 studied SNPs, which indicates that the studied SNPs loci are not linked to be inherited together [43].The results also suggested that the TGC, CGC, and CGG haplotypes are risk factors for HCC, while the CGG and TTG haplotypes increase the chance of LC turning into HCC.
There is a long list of risk factors for chronic hepatitis patients' condition progressing to HCC.In determining HCC risk, smoking seems to correlate with HBV and HCV [42].HCC is also known to be more common in men, and numerous SNPs have been linked to a higher genetic susceptibility to the disease [44].In this study, there was no significant association between VDR SNPs (rs2228570 and rs3782905) and smoking status in HCC risk, which agreed with Galal et al., [24].However, the GG genotype of DBP (rs7041) SNP was significantly associated with disease progression in both groups (non-smokers and smokers).By looking at gender, in males, the GC genotype of the rs3782905 SNP might be a risk factor for HCC.On the other hand, the GG genotype of rs7041 SNP was significantly associated with increasing LC to HCC in females.Our results were in the same line with Chuang et al. [44] and Yang et al. [45].

Conclusion
The current study provided evidence that among the four studied SNPs, rs3782905 and rs7041 might be viewed as risk factors for either the progression of chronic HCV liver disease into HCC in Egyptian patients.Up to our knowledge, this is the first study to include the genetic variant of DBP in association to the HCV infected LC and HCC Egyptian patients.In addition, we highlighted the role of VDR and DBP SNPs with smoking status as well as gender in HCC susceptibility.We recommend screening of these SNPs for prediction of LC and HCC development in HCV infected patients, which may improve the used therapeutic protocol.These results suggest that VDR polymorphisms may be potential determinants for HCC susceptibility in Egyptian HCV patients.Future research with larger sample size of subjects with HCV infection is advised, because chronic liver disease induced by HCV infection is the primary cause of HCC in Egypt.

Table 2
Demographic and biochemical characteristics of all studied groups

Table 3
Association analysis ofgenotype distributions and alleles frequencies of the VDR and DBP SNPs

Table 4
Comparison of all groups under study's VDR and DBP gene polymorphisms' association haplotypes p p-value is significant < 0.05, NS Non-significante

Table 5
Smoking status and SNPs of VDR and DBP gene association analyses in cirrhotic and HCC patients

Table 6
Gender and SNPs of VDR and DBP geneassociation analyses in cirrhotic and HCC patients